Pyc Therapeutics Limited · ASX:PYC
Pyc Therapeutics — Phylogica — Conference Presentation (July 2007)
50 slides from Pyc Therapeutics's conference presentation, Jul 11, 2007
Slide 1: Phylomers: a new class of peptide derived from protein fragments
For personal use only Phylomers: a new class of peptide derived from protein fragments suitable for intracellular and extracellular targets Paul Watt, VP Drug Discovery www.phylogica.com
Slide 2: Summary of key points
Summary of key points a new approach for therapy and target validation For personal use only Nature uses protein fragments and multiple protein fragment-derived therapeutics are already marketed and in the clinic What are Phylomers? Advantages Hit rates from Phylomer library screens In vitro validat
Slide 3: Natural protein fragments as functional peptides
Natural protein fragments as functional peptides For personal use only Insulin from pre-pro-insulin and pro-insulin Antimicrobial peptides in milk are proteolytic fragment of lactoferrin The peptide hormones ACTH, CLIP and MSH derived from a single precursor protein A peptide angiogenesis inhibitor
Slide 4: Protein fragment drugs: an emerging new class
Protein fragment drugs: an emerging new class For personal use only Teriparatide (Forteo™, Annual sales of approximately US$600M) a recombinant 34aa N-terminal fragment of human parathyroid hormone Enfuvirtide (Fuzeon™;Trimeris/Roche, Annual sales more than US$150M). Interfacial 36aa peptide from HI
Slide 5: Phylomer Peptides
Phylomer® Peptides a diverse class of peptides from natural proteins For personal use only Phylomers® represent a new class of peptides derived from natural protein fragments encoded by biodiverse ancient bacterial genomes From 25 characterised bacterial genomes we have derived 260 million distinct
Slide 6: Benefits of Phylomer-based therapeutics
Benefits of Phylomer®- based therapeutics Phylomer® peptides can offer significant advantages For personal use only beyond current antibody therapies; - Able to hit Intracellular targets - Reduced cost of goods - No antibody intellectual property royalty stack - Alternative patient friendly delivery
Slide 7: Approaches to Phylomer screening
Approaches to Phylomer® screening For personal use only Intracellular target screens: Forward two hybrid Reverse two hybrid Extracellular target screens: Lytic Phage Display (T7) Secreted Phage Display (M13) Phenotype driven screens: Synthetic Phylomer library (in development) Array applications Par
Slide 8: Phylomer screen statistics show high hit-rates
Phylomer® screen statistics show high hit-rates yeast two-hybrid screen against an intracellular target For personal use only More than 50% of the hits show biological activity in preliminary ex vivo cell- based reporter assays Significant enrichment for natural ORFs • <15% of library is in natural
Slide 9: Extracellular targets program
For personal use only Extracellular targets program: screening Phylomers® to inhibit the function of targets that play a crucial role in inflammation
Slide 10: Screening against extracellular targets
Screening against extracellular targets validated therapeutic targets for inflammation For personal use only TNFα ligand (antagonist) CD40 ligand (antagonist) CD28 receptor (antagonist) GM-CSF ligand (antagonist)
Slide 11: Phylomer hits against extracellular targets
Phylomer® hits against extracellular targets phage display identifies target-specific hits For personal use only Phage display screens: assessment of relative binding and specificity using Phage ELISA BSA control TNF-α CD40L
Slide 12: High-throughput screens for high affinity hits
High-throughput screens for high affinity hits spot competition ELISA assay For personal use only Immobilised target competes for binding to phage-displayed Phylomer® peptide in the presence of 0.5 µM and 1 µM free target 48 Phylomer® hits gainst CD40L with IC50s of under 500nM
Slide 13: Phylomer affinities
Phylomer® affinities low nanomolar IC50 values with naïve Phylomers® For personal use only Assessment of relative binding affinity using competition ELISA Immobilised target competes for binding to phage-displayed peptide in the presence of synthesised free Phylomer® peptide (26 aa) or free target H
Slide 14: Intracellular targets program
For personal use only Intracellular targets program: screening Phylomers® to inhibit the function of targets that play a crucial role in inflammation: AP1, NFκB and a TLR signalling adaptor
Slide 15: Screening Phylomer peptides against intracellular target
Screening Phylomer® peptides against intracellular target For personal use only AP-1 is one of the best characterised mammalian transcription factors Activation of the c-Jun/JNK pathway has been validated as critical in the acute stress response such as: • Cell death immediately following cardiac is
Slide 16: Brain Damage in Stroke
Brain Damage in Stroke ischemia is followed by apoptotic damage For personal use only Long-term apoptosis is a significant cause of enduring brain damage After a stroke the cells surrounding the initial lesion die over hours to days No effective treatment exists to prevent this neuronal cell death a
Slide 17: Acute ischemic brain damage
Acute ischemic brain damage inhibiting AP-1 activity For personal use only Obstruction to arterial blood supply Cell membrane depolarisation ? JNK ? Jun Jun Initial apoptosis of Rapid necrotic Late stage neurons neuronal death apoptosis of neurons
Slide 18: Screening Phylomer peptides to disrupt AP-1 signalling
Screening Phylomer® peptides to disrupt AP-1 signalling Summary For personal use only Yeast-two hybrid Hits to disrupt AP-1 Notes screening pathway No. of Clones screened 300,000 This represented a minor fraction of the Phylomer library No. validated primary hits 60 Confirmed from 90 hits by recapit
Slide 19: Structures of Phylomer hits
Structures of Phylomer® hits modeling onto parent protein Phylomer® peptides contain a variety of structural motifs, such as single For personal use only helices, helix-turn-helix, strand-turn-strand Modeling suggest some Phylomer® sequences could form stable tertiary structures in solution Phylomer
Slide 20: Validating an intracellular target: blocking AP1 using Phylomers
For personal use only Validating an intracellular target: blocking activity of the transcription factor AP1 using Phylomers®
Slide 21: Phylomer efficacy in vitro
Phylomer® efficacy in vitro neuronal injury models For personal use only Glutamate excitotoxicity model NMDA excitotoxicity model Transient oxygen-glucose deprivation model
Slide 22: Glutamate excitotoxicity model
Glutamate excitotoxicity model protection against glutamate-induced apoptosis For personal use only Exposure Measure Synthesised to 100µM Culture neuronal peptide glutamate at 37°C survival Rat Ratneuronal neuronalcells cells 15 min 5 min 24 hours
Slide 23: Synthetic Phylomers protect neuronal cells
Synthetic Phylomers® protect neuronal cells multiple Phylomer® hits show significant neuroprotection For personal use only 100 ** p < 0.0001 ** % Neuronal viability 80 ** ** 60 ** 40 ** ** 20 0 L-PYC19-TAT L-PYC35-TAT L-PYC36-TAT L-PYC38-TAT L-PYC41-TAT No insult TAT-D No peptide D-JNKI-1 -TAT
Slide 24: Dose-dependent neuroprotection
Dose-dependent neuroprotection L & D Phylomers® inhibit glutamate-induced apoptosis For personal use only PYC35 PYC36
Slide 25: NMDA excitotoxicity model
NMDA excitotoxicity model protection against NMDA-induced apoptosis For personal use only Exposure Measure Synthesised Culture to 200µM neuronal peptide at 37°C NMDA survival Ratneuronal Rat neuronalcells cells 15 min 15 min 24 hours
Slide 26: Phylomers confer neuroprotection
Phylomers® confer neuroprotection inhibition of NMDA-induced apoptosis For personal use only p < 0.0001 % Neuronal survival 100 80 60 40 20 0 No peptide Blockers No insult PYC35-TAT PYC36-TAT
Slide 27: Transient oxygen-glucose deprivation model
Transient oxygen-glucose deprivation model mimicking conditions following cerebral ischemia For personal use only Pre-insult Post-insult Synthesised Synthesised peptide peptide Wash; Re-supply Incubation in Anaerobic culture Measure Glucose-free/ incubator medium; neuronal Deoxyglucose Glucose/ surv
Slide 28: Protection from ischemia-induced death
Protection from ischemia-induced death inhibiting cell death pre-and post oxygen-glucose deprivation For personal use only p < 0.0001 % Neuronal survival 100 80 60 40 20 0 No peptide No insult Post-insult Post-insult Pre-insult Pre-insult D-PYC35-TAT D-PYC36-TAT
Slide 29: Pharmacological properties of Phylomers
For personal use only Pharmacological properties of Phylomers®: stability, pharmacokinetics, and immunogenicity
Slide 30: Stability of Phylomers
Stability of Phylomers® high stability of modified and unmodified peptides For personal use only Plasma stability of unmodified lead Phylomers® range from 10 minutes to 30 minutes in rat plasma - Unmodified Phylomers® are stable in buffer, but are degraded by proteases in plasma as with many other p
Slide 31: In vitro stability
In vitro stability Phylomer® half-life in human plasma For personal use only Quantitation of Phylomer® peptides using High Performance Liquid Chromatography (HPLC) and Mass Spectrometry (MS) for the duration of the study (up to 12 hrs) Phylomer® [µg/ml] Time [min] InSymbiosis, Canada
Slide 32: In vivo stability
In vivo stability pharmacokinetic studies in rats For personal use only Quantitation of Phylomer® peptides levels by HPLC/MS in blood samples over a period of time following intravenous injection In vivo half-lives of 35-100 minutes for unPEGylated Phylomer® peptides • Half life of PEGylated phylome
Slide 33: Immunogenicity potential of Phylomers
Immunogenicity potential of Phylomers® in silico and ex vivo approaches to assess immunogenic potential For personal use only In silico testing of 28 Phylomer® peptides tested against multiple HLA haplotypes - Only a few Phylomers® have a high chance of being immunogenic Phylomer® hits against AP-1
Slide 34: Modeling Phylomer immunogenicity
Modeling Phylomer® immunogenicity low immunogenic potential For personal use only Calcitonin (32) r-Insulin (51) Fuzeon (36) Phylomer® peptides (14-80 aa) +++ MHC Ligands/Peptide ++ + s y pe e t id llo ept A /p HC nd M ou 1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 b Peptide No. Accuro Biologics Ltd,
Slide 35: In vivo efficacy: stroke, head trauma and burns
For personal use only In vivo efficacy: assessing Phylomers® for treatment of stroke, head trauma and burns
Slide 36: Phylomer efficacy in vivo
Phylomer® efficacy in vivo models of head trauma, stroke and burns For personal use only Global model of ischemic stroke Focal cortical model of CNS injury Full thickness burns model Inhaled LPS model of ARDS
Slide 37: Global model of ischemic stroke
Global model of ischemic stroke In vivo rat model of cerebral ischemia For personal use only Rat two vessel occlusion with hypotension model: both carotid arteries are occluded lowering the blood pressure to 45mmHg, followed by reperfusion and restoration of blood pressure 11nmoles Global peptide As
Slide 38: Neuroprotection in a global ischemia model
Neuroprotection in a global ischemia model post insult treatment with Phylomers® For personal use only Hippocampal CA1 Cell Count 350 n=3 *p<0.05 compared with NaCl 300 250 200 n=6 150 * 100 n=3 50 n=5 0 Sham NaCl PYC36 D-PYC36 scram 8 minute global cerebral ischaemia 11nmoles peptide (ICV) added 1
Slide 39: Focal model of cortical CNS injury
Focal model of cortical CNS injury in vivo rat model of traumatic brain injury For personal use only There are currently no effective treatment available for Traumatic Brain injury (TBI); e.g. invasive head injuries Similar pathways of neuronal cell death are activated in the case of stroke and TBI
Slide 40: Protection from neuronal death
Protection from neuronal death significantly reduced cortical lesions For personal use only Extent of neuronal death measured as loss of MAP immunoreactivity in representative cortical lesions 24 hrs post injury and concomitant treatment with Phylomers® 2.5 Loss of MAP2 (mm2) 2 1.5 1 * 0.5 0 D-PYC35
Slide 41: Phylomer efficacy in vitro and in vivo: skin burns
Phylomer® efficacy in vitro and in vivo Skin burns (apoptosis and wound healing models) For personal use only Immediately following a burn injury, the cells surrounding the injury site undergo cell death which inhibits wound healing and increases scaring (first 12 hours critical) Phylomer treatment
Slide 42: Phylomer Efficacy: In-vitro apoptotic assays
Phylomer® Efficacy: In-vitro Apoptotic assays For personal use only UV Irradiation 2hr incubation Add Annexin V Ab Keratinocytes Peptide added and PI and analyse (Skin Cells) using FACS Using Annexin V and Propidium Iodide double labelling and FACS analysis of cell populations to determine percentag
Slide 43: Apoptotic assays human keratinocytes
Phylomer® Efficacy: In-vitro Apoptotic assays human keratinocytes For personal use only * Statistically significant p< 0.05
Slide 44: Full thickness burns model
Full thickness burns model in vivo mouse model of burn injuries For personal use only Burn injuries are the third leading cause of accidental death in the US After a burn injury, the cells surrounding the injury site undergo cell death similar to the damage following a stroke 1) Full-thickness burn
Slide 45: Histology of burn site
Histology of burn site mouse full thickness burns model For personal use only Day 1 Control 2x Control 20x PYC35B Treated 2x Treated 20x
Slide 46: Treatment of burns with Phylomers
Intranasal Treatment of burns with Phylomers® topical application of Phylomers® improves wound healing For personal use only Treated wounds close faster: from day 7, treated burns have significantly more re-epithelialised and the distance between the wound edges is significantly smaller Day 11 Reduc
Slide 47: Treatment of ARDS with Phylomers
Treatment of ARDS with Phylomers® Animals were either untreated (control), given LPS alone (open bars), given LPS and treated For personal use only with scrambled peptide (filled bar), or treated with AP-1 blocking peptides (hatched bars) and BAL performed 6 hour post LPS administration. Treatment w
Slide 48: Phylomer peptides as target validation tools
Phylomer peptides as target validation tools Acting at the level of protein interactions For personal use only Watt, PM et al., (2006) Expert Opin. Drug Discov. 1:492-502
Slide 49: Summary: Phylomers
Summary: Phylomers Peptides from parts of biodiverse proteins For personal use only Phylomer advantages: - Can be Synthesised (Reduced costs) - Alternative delivery potential (Patient Friendly) inhaled, buccal, transdermal, nasal,oral delivery routes - No antibody-associated royalty stack - Can be u
Slide 50: Phylogica's team, collaborators & co-development partners
Phylogica’s team, collaborators & co-development partners Paul Watt Marie Scobie Mark Fear For personal use only Nadia Milech Daniel Shaw Vanessa Cull Richard Hopkins Clinton Hall Katharine Adcroft Michelle Schaffer Scott Winslow Ryan Jeffries Darryl D’Souza Patrick Holt Darcelle Dixon Deborah Stric